Protein phosphorylation regulates virtually all cellular processes and aberrant phosphorylation is the underlying cause of numerous cancers. The majority of intracellular proteins are phosphorylated at any given time, and, while 9 of the 20 amino acids can be phosphorylated, Ser, Thr, and Tyr phosphorylation has attracted the majority of attention in eukaryotic organisms. Histidine (His) phosphorylation has long been implicated in signal transduction (e.g. prokaryotic “two-component” systems); however, its role in mammalian cells remains largely unexplored. This is due to the difficulty in studying His phosphorylation by standard biochemical techniques.
Unlike pTyr, pSer and pThr, phosphohistidine (pHis) is heat and acid labile. Consequently, the importance of His phosphorylation has been greatly underestimated. Despite the brief half-life of pHis under acidic conditions (18-25 sec half-life in 1 M HCl, 49° C.), the high-energy, phosphoramidate bond of pHis is stabilized by basic conditions, and the half-life in protein substrates is strongly influenced by neighboring amino acid residues indicating it is highly context dependent (12 day half-life of histone H4 at RT, pH 7.6). Thus, new tools are needed to bring this under-appreciated post-translational modification to light.
Despite the paucity of knowledge about His phosphorylation in eukaryotic signal transduction, there is growing evidence implicating His kinases in cancer and tumor metastasis. In fact, the first metastasis suppressor gene identified (by its reduced expression in highly metastatic melanoma cell lines) is one of only two known mammalian His kinases; Nm23-H1 (AKA NME1 or nucleoside diphosphate kinase [NDPK-A]). Nm23 family members are involved in intracellular nucleotide homeostasis as well as in both physiological and pathophysiological cellular processes such as proliferation, differentiation, development, apoptosis, cytokinesis and metastasis, through mechanisms that remain largely unknown.
Nm23-H1 and the closely related Nm23-H2 (NME2/NDPK-B) catalyze transfer of phosphate from ATP onto nucleoside diphosphates (NDPs) through a pHis enzyme intermediate. Nm23-H1/-H2 also possess His kinase activity, transferring the phosphate from the active site pHis onto a His in a target protein. However, the lack of pHis-specific antibodies (Abs) and pHis's instability under typical conditions used for proteomics have made it difficult to study the role His phosphorylation plays in suppression of metastasis. Phosphospecific Abs exist for pSer, pThr and pTyr, and these, combined with biochemical and proteomic techniques, have proved invaluable in the study of protein phosphorylation in cellular signaling and cancer. Until recently, the difficulties in creating stable pHis peptides have precluded generation of pHis specific Abs. Development of non-cleavable pHis analogues now makes this possible [Kee et al., (2010) J Am Chem Soc. 132, 14327-9 and McAllister et al., (2011) Chem Commun. 47, 1297-9]. These pHis analogues will be used as immunogens to make Abs specific for both biologically relevant pHis isomers, 1- and 3-pHis (FIG. 1). Anti-1- and 3-pHis Abs will serve as novel tools to study His kinase activity of Nm23 proteins as well as yet undiscovered His kinases and their substrates.
It has been estimated that 6% of phosphorylation in eukaryotes occurs on His and that pHis could be 10-100 times more abundant than pTyr. Despite this, only a handful of mammalian pHis proteins, two His kinases (Nm23-H1/-H2) and a single pHis phosphatase (PHPT1) have been identified. For the few known pHis substrates, this phosphorylation has proved essential to their function and revealed novel signaling pathways. Nm23-H2 phosphorylates KCa3.1 (H358) and is required for potassium channel activation. Phosphorylation of heterotrimeric Gs protein subunit β1 (H266) by Nm23-H2 activates Gs and regulates basal cAMP accumulation. Furthermore, Nm23-H2, G proteins and caveolin expression are mutually dependent for stable localization and caveolae formation. Histone H4 phosphorylation (H18) is associated with enhanced cell proliferation in liver and thymus. The development of pHis-specific Abs combined with improved techniques for pHis peptide enrichment and identification by MS will be used to greatly expand the number of known pHis targets and determine which ones play a role in suppression of tumor metastasis by His kinases. In addition, this protocol may identify novel His kinases, since known His kinases autophosphorylate on His.